Cortisol In Urine Samples Research Articles

MEASUREMENT OF CORTISOL IN HUMAN PLASMA AND URINE BY ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Objective: The objective of this study is to develop and validate a simple, sensitive, specific, and rapid assay for quantification of clinically relevant cortisol level in human plasma and urine samples.Methods: Ultra performance liquid chromatographic-tandem mass spectrometric (UPLC-MS/MS) analysis was performed on Atlantis dC18 column (2.1×100 mm, 3 μm) with a mobile phase consisting of acetonitrile and 2 mM ammonium acetate (50:50, v: v) that was delivered at a flow rate of 0.3 ml/min. Tolperisone (2 ng) was used as an internal standard (IS). Biological samples were extracted with a mixture of hexane and methyl tert-butyl ether (8:2, v: v). The eluents were monitored using electrospray ionization in the positive ion mode with transition mass to charge ratio set at 363.1 → 121.0 and 246.0 → 97.9 for cortisol and IS, respectively. The method was validated according to international guidelines.Results: Retention times of cortisol and IS were about 1.4 and 2.3, respectively. Relationship between cortisol level and peak area ratio of cortisol to IS was linear (R2≥0.987) in the range of 2.5–400 ng/ml and 1.0–200 ng/ml in plasma and urine samples, respectively. Intra- and inter-day coefficient of variation and bias were ≤9.7% and ±11.1%, and ≤10.4% and ±11.5% for plasma and urine samples, respectively. Extraction recoveries were in 80–91% for cortisol plasma and 80–86% for cortisol in urine samples. Cortisol was ≥86% stable when stored at room temperature for 24 h or at −20°C for 24 weeks in plasma samples and ≥93% stable when stored at room temperature for 24 h or −20°C for 16 weeks in urine samples.Conclusion: We report a validated, clinically relevant, simple, and rapid UPLC-MS/MS assay of cortisol level in human plasma and urine.

Specific gravity and creatinine as corrections for variation in urine concentration in humans, gorillas, and woolly monkeys

Hormones excreted in the urine are widely used to assess the physiological and psychological condition of unrestrained animals. In order to control for variation in the water concentration of urine samples, the hormone concentration is often indexed to the concentration of creatinine. Because there are several problems with using creatinine, we have investigated the efficacy of specific gravity as an alternative basis for adjusting the hormone concentration in humans, gorillas, and woolly monkeys. In an experimental manipulation of human urine hydration, ten volunteers drank a water load proportional to body weight, and provided complete urine collection and saliva samples for four consecutive 20 min intervals. From the urine, we measured cortisol (radioimmunoassay), creatinine (colorimetric assay), and specific gravity (refractometer). Only cortisol was assayed from saliva. During 80 min following water ingestion, cortisol, creatinine, and specific gravity declined as urine became diluted; however, total cortisol excretion remained constant. Only cortisol concentration indexed to specific gravity accurately reflected the consistent cortisol excretion. Specific gravity and creatinine-corrected cortisol values were highly correlated but were significantly different. Salivary cortisol provided evidence for the relative stability of serum cortisol. To determine the utility of these corrections in other primates, we compared specific gravity- and creatinine-corrected cortisol in urine samples from captive gorillas (N=16) and woolly monkeys (N=8). As with the human study, the two corrections were strongly correlated in each species, but the means were different. Specific gravity correction was superior in revealing the circadian variation in cortisol.

Micellar electrokinetic chromatography for the determination of cortisol in urine samples in view of biomedical studies

An MEKC method used for the determination of cortisol in urine was developed and elaborated. In turn, the measurements of urinary free cortisol provided the diagnostic information for excess adrenal production of cortisol. MEKC realized by the addition of anionic surfactant SDS to the buffer solution was demonstrated to be the appropriate mode for the separation of cortisol and dexamethasone was used as internal standard. A buffer solution composed of 10 mM sodium tetraborate and 50 mM SDS at pH 8.8 was used. The MEKC assay was evaluated by analyzing a series of urine samples containing cortisol in variable concentrations. The proposed method was validated for specificity, linearity, LODs and LOQs, precision and trueness. The LOQ for cortisol equaled 5 ng/mL. The method was selective and reliable for identification and can detect changes of endogenous levels of cortisol in urine under different stress situations.

Convenient chromatographic prepurification step before measurement of urinary cortisol by radioimmunoassay

We applied various prepurification protocols (extraction with different solvents, liquid/solid separation on bonded silica media, Celite, and Sephadex LH20 chromatography) with a range of commercially available RIA kits to measure cortisol in urine samples. We then compared the results with the concentrations measured by a HPLC method validated with reference to isotope dilution gas chromatography-mass spectrometry. We conclude that chromatography on a commercial, prepacked diol minicolumn (Waters Sep-Pak Vac RC) in combination with dichloromethane extraction is a convenient and very effective purification step before RIA of urinary cortisol in patients not receiving corticoid medication. We tested numerous steroids for interference and found that free polar cortisol derivatives (hydroxylated or hydrogenated) could only partially account for the overestimations routinely encountered when free urinary cortisol concentrations are measured by direct RIA.